ES2635691T3 - Use of relaxin to increase arterial compliance - Google Patents

Abstract

Relaxin H2 for use in a method to increase arterial compliance in a human being, where the subject has a decrease in global arterial compliance in relation to the overall arterial compliance in a healthy subject, where the subject has one or more selected conditions from the group consisting of: atherosclerosis, coronary heart disease, diastolic dysfunction, hypercholesterolemia, left ventricular hypertrophy, arterial stiffness associated with long-standing smoking, arterial stiffness associated with obesity, arterial stiffness associated with age and systemic lupus erythematosus.

Description

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Use of relaxin to increase arterial compliance

5 Description

FIELD OF THE INVENTION

[0001] The present invention is in the field of arterial compliance, and in particular, the use of relaxin to increase arterial compliance.

BACKGROUND OF THE INVENTION

[0002] Arterial compliance declines with age even in healthy individuals without cardiovascular disease

15 manifests. With age, there is a decrease in the ability of large and small arteries to distend in response to an increase in pressure. The reduction associated with age in arterial compliance is an independent risk factor for the development of cardiovascular disease, and is associated with other pathological conditions. For example, reduced arterial compliance is also associated with type 1 diabetes and type 2 diabetes. Diabetic arteries have been reported to appear to age at an accelerated rate compared to

20 arteries of non-diabetic individuals. See, eg, Arnett et al. (1994) Am J Epidemiol. 140: 669-682; Rowe (1987) Am J Cardiol. 60: 68G-71G; Cameron et al. (2003) Diabetes Car. 26 (7): 2133-8; Kass et al. (2001) Circulation 104: 1464-1470; Avolio et al. (1983) Circulation 68: 50-58; U.S. Patent No. 6,251,863; U.S. Patent No. 6,211,147.

[0003] There is a need for methods of increasing arterial compliance, and for treating disorders associated with or resulting from reduced arterial compliance. The present invention addresses these needs.

BRIEF DESCRIPTION OF THE INVENTION

[0004] The present invention shows the use, to treat individuals with decreased compliance

30, of an effective amount of a formulation comprising a relaxin receptor agonist, which is a recombinant human relaxin, that is, human H2 relaxin.

[0005] In a context of the invention, the invention provides a method for increasing arterial compliance in a subject, wherein said method comprises measuring the overall arterial compliance in a subject; determine that the overall arterial distensibility is decreased in the subject in relation to the global arterial distensibility in a healthy subject; and administering to a subject a pharmaceutical formulation comprising relaxin to increase arterial compliance in the subject. The overall arterial compliance can be measured, in a context, from the diastolic decay of the aortic pressure wave using the area method. In another context, global arterial distensibility can be calculated as the volume beat beat ratio to pulse pressure, where the beat volume is

40 defines as the ratio of cardiac output to heart rate.

[0006] In related contexts, the local arterial distensibility or regional arterial distensibility of a subject can be measured in addition or as an alternative to the measurement of global arterial distensibility and, if the global or regional arterial distensibility decreases in relation to arterial distensibility expected global or regional

45 for a healthy individual in a similar situation, relaxin can be administered to increase arterial compliance in that individual.

[0007] In other contexts, the subject to whom relaxin is administered suffers one or more of the following disorders: atherosclerous, coronary heart disease, diastolic dysfunction, hypercholesterolemia, left ventricular hypertrophy stiffness

50 arterial associated with long-term smoking, arterial stiffness associated with obesity, arterial stiffness associated with age, systemic lupus erythematosus and hypercholesterolemia. In related contexts, the invention provides methods for increasing arterial compliance in perimenopausal, menopausal and postmenopausal women and in individuals who are at risk of one of the aforementioned disorders.

[0008] In a further context of the invention, the administration of relaxin increases arterial distensibility by at least 10%, 15%, 20% or more, in relation to arterial distensibility measured before administration. In other additional contexts, the invention provides for the administration of relaxin to individuals with decreased arterial compliance at a predetermined rate in order to maintain a serum relaxin concentration of 0.5 to 80 ng / ml. In a context, relaxin is recombinant human relaxin. In another context, the

60 relaxin is recombinant H2 relaxin. In related contexts, relaxin can be administered daily, in an injectable formulation, as a sustained release formulation or as a continuous infusion.

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BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

5 Figures 1A-C show in percentage change from the start for cardiac output (Figure 1A), heart rate (Figure 1B) and volume beat (Figure 1C) in female rats given recombinant human relaxin at low doses (rhRLX; 4 µg / h), high dose rhRLX (25 µg / h) or vehicle.

Figures 2A-D show the percentage change from the start for systemic vascular resistance (Figure

10 2A), mean blood pressure (Figure 2B), global blood distensibility (Figure 2C) and pulse rate pulse volume ratio in female rats given low doses of rhRLX, high doses of rhRLX or vehicle.

Figures 3A and 3B show the blood pressure traces representative of a rat (Figure 3A); Y

15 assemble the mean blood pressure waves for the three groups (vehicle, low dose rhRLX and high dose rhRLX) on day 10 after implantation of the osmotic minipump (Figure 3B).

Figures 4A and 4B show circumferential stress () -radio of the middle wall (Rm) (Figure 4A); and the incremental elastic modulus (Einc) -Rm ratios (Figure 4B) for small renal arteries isolated from

20 rats treated with rhRLX or vehicle for 5 days.

Figure 5 shows temporary changes in systemic hemodynamics in response to the administration of a low dose (4 µg / h) of recombinant human relaxin in male and female rats. Data for heart rate (A), heartbeat volume (B), cardiac output (C) and mean blood pressure (D) are

25 present as percentages of the initials. * P <0.05 vs. baseline (Fisher's LSD post hoc). Significant increases in SV are shown only for days 6, 8 and 10.

Figure 6 shows the temporary changes in systemic arterial properties in response to the administration of a low dose (4 µg / h) of recombinant human relaxin in male and female rats. The

30 data on systemic vascular resistance (A) and two measures of global arterial compliance, (ACarea (B) and SV / PP (C), are presented as percentages of the initials. * P <0.05 vs. initial (Fisher's LSD post -hoc) Significant increases in ACarea and SV / PP are shown only for days 8 and 10.

Figure 7 shows temporary changes in systemic hemodynamics in response to the administration of

35 three doses of recombinant human relaxin in female rats: low (4 µg / h), medium (25 µg / h) and high (50 µg / h). Data for heart rate (A), heartbeat volume (B), cardiac output (C) and mean blood pressure

(D) are presented as percentages of the initials. * P <0.05 vs. baseline (Fisher LSD post-hoc).

Figure 8 shows temporary changes in systemic hemodynamics in response to the administration of

40 three doses of recombinant human relaxin in female rats: low (4 µg / h), medium (25 µg / h) and high (50 µg / h). The data of systemic vascular resistance (A) and two measures of global arterial compliance, ACárea (B) and SV / PP (C), are presented as percentages of the initials. * P <0.05 vs. baseline (LSD of

Fisher post hoc).

45 Figure 9 shows the temporary changes in systemic hemodynamics in response to the administration of the short-term high dose of recombinant human relaxin in female rats. Heart rate (A), heartbeat volume (B), cardiac output (C) and mean blood pressure (D) data are presented as percentages of the initials. * P <0.05 vs. baseline (Fisher's LSD post hoc). Figure 10 shows the temporary changes in systemic arterial properties in response to the administration of high doses at short

50 term recombinant human relaxin in female rats. The data of systemic vascular resistance (A) and two measures of global arterial compliance (Acarea (B) and SV / PP (C)) are presented as percentages of the initials.

Figure 11 shows the relationships between the percentage changes made from the beginning and the values

55 initials for systemic vascular resistance (A) and two measures of global arterial distensibility, ACarea (B) and SV / PP (C), in male and female rats given low doses of recombinant human relaxin (4 µg / h). These relationships were independent of sex. The solid line in each panel corresponds to the graph of the relationship obtained by linear regression (male and female rats combined).

60 DEFINITIONS

[0010] The terms "subject," "host," "individual," and "patient," used interchangeably herein, refer to any subject, particularly a mammal, for which a diagnosis or therapy is desired, particularly humans. . Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats,

65 mice, horses, etc. In many contexts, a subject is a human in need of treatment for a disease or condition related to or resulting from reduced arterial compliance.

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[0011] The terms "treatment", "treating", "therapy", etc., are used herein to generally refer to obtaining a desired therapeutic, pharmacological or physiological effect. The effect can be prophylactic in terms of preventing

5 fully or partially a disease or symptoms and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, eg, a human being, and includes: (a) preventing the disease from occurring in a subject that may be predisposed to it, but to whom He has not been diagnosed yet; (b) inhibit the disease, that is, stop its development; and (c) alleviate the disease, that is, cause disease regression.

[0012] As used herein the terms "isolated" and "substantially purified", used interchangeably, when used in the context of "isolated relaxin", refers to the relaxin polypeptide which is a different environment from that in the which naturally appears the relaxin polypeptide. As used herein, the term "substantially

"Purified" refers to a relaxin polypeptide that is removed from a natural environment and is at least 60% free, preferably 75% free, and more preferably 90% free from other components with which it is naturally associated.

[0013] Before describing the present invention, it should be understood that this invention is not limited to the particular contexts described. The scope of the present invention will be limited only by the statements included.

[0014] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lowest limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any value established or intervening in that established range, is included within the invention.

The upper and lower limits of these minor ranges may be included independently in the minor ranges, and are also included within the invention, subject to any limits specifically excluded in the established range. Where the established range includes one or both limits, the ranges that exclude either of them or both included limits are also included in the invention.

[0015] Unless otherwise defined, all technical and scientific terms used here have the same meaning commonly understood by a person skilled in the art to which this invention belongs. Although methods and materials used or equivalent to those described herein can also be used in the practice or evaluation of the present invention, preferred methods and materials are now described.

35 [0016] It should be noted that when used here and in the declarations included, the singular forms "a", "and" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the reference to "a relaxin formulation" includes a plurality of such formulations and the reference to "the active agent" includes the reference to one or more active and equivalent agents known to those skilled in the art, and thus successively.

[0017] The publications discussed here are provided for disclosure only before the date of registration of this application. Nothing here will be constructed as an admission that the present invention cannot precede such publication by virtue of a prior invention. Thus, the publication dates provided may be different from the actual publication dates that may have to be independently confirmed.

45 DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides methods for treating disorders associated with arterial stiffness; methods to increase arterial compliance; methods to reduce arterial stiffness in an individual; and methods to reduce the risk that an individual develops one or more complications or disorders associated with a reduction in arterial compliance. The methods generally involve administering to an individual in need thereof an effective amount of a relaxin receptor agonist. In some contexts, the individual has, or is at risk of developing, age-related arterial stiffness. In other contexts, the individual is a perimenopausal, menopausal or postmenopausal woman, and thus has developed, or is at risk of developing, stiffness.

55 arterial.

[0019] One of the greatest cardiovascular adaptations in human pregnancy is the increase in global arterial compliance, accompanied by increases in relaxin levels, which reach a peak at the end of the first trimester just when systemic vascular resistance (SVR) It reaches a nadir. At least in theory, the increase in global arterial compliance is critical for the maintenance of cardiovascular homeostasis during pregnancy for several reasons: (1) the increase in CA prevents excessive decrease in diastolic pressure which would otherwise fall at precariously low levels due to the significant decrease in SVR; (2) the increase minimizes the pulsatile or oscillatory work performed by the heart which would otherwise increase in disproportion to the increase in the total work required and invested by the heart during pregnancy; and (3) the increase in global CA preserves the stress of continuous friction (or avoids oscillatory friction) in the blood-endothelial interface despite the hyperdynamic circulation of pregnancy, thus favoring the production of

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nitric oxide instead of superoxide and other reactive oxygen species harmful by the endothelium. The increase in global CA, together with the reduction in SVR, can result in a circulatory deficit, and thus contribute to the retention of sodium and water and the expansion of plasma volume during the beginning of pregnancy.

5 METHODS OF TREATMENT

[0020] The present invention provides methods for increasing arterial compliance which use the step of administering to an individual in need thereof an effective amount of a relaxin receptor agonist. In some contexts, the individual has atherosclerosis, coronary heart disease, diastolic dysfunction, familial hypercholesterolemia, left ventricular hypertrophy, arterial stiffness associated with long-term smoking, arterial stiffness associated with obesity arterial stiffness associated with age, systemic lupus erythematosus and hypercholesterolemia, or is at risk of developing age-related arterial stiffness. In other contexts, the individual is a perimenopausal, menopausal or postmenopausal woman, or a woman who has stopped her periods for reasons not related to age, e.g., due to excessive exercise or as a result of surgery (e.g. ., hysterectomy,

15 oophorectomy). and has developed, or is at risk of developing, arterial stiffness.

[0021] The methods generally involve administering to an individual an effective amount of relaxin. In some contexts, an effective amount of relaxin is an amount that is effective in increasing arterial compliance at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%, or more, compared to arterial compliance in the absence of relaxin treatment.

[0022] In some contexts, an effective amount of relaxin is an amount that is effective in reducing arterial stiffness by at least 5%, at least 10%, at least 15%, at least 20%, at least in 25% at least

25 in 30%, at least 35%, at least 40%, at least 45% or at least 50%, or more, compared to arterial stiffness in the individual in the absence of relaxin treatment.

[0023] In some contexts, an effective amount of relaxin is an amount that is effective in reducing arterial stiffness by at least 5%, at least 10%, at least 15%, at least 20%, at least in 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%, or more, compared to arterial stiffness in the individual in the absence of relaxin treatment .

[0024] Disorders resulting from or associated with arterial stiffness or reduced arterial compliance include, but are not limited to, atherosclerosis, coronary heart disease, diastolic dysfunction, familial hypercholesterolemia, hypertrophy

35 left ventricular, arterial stiffness associated with long-standing smoking, arterial stiffness associated with obesity, arterial stiffness associated with age, systemic lupus erythematosus and hypercholesterolemia. Of particular interest in some contexts is arterial stiffness associated with normal aging, diastolic dysfunction, menopause, obesity, hypercholesterolemia, familial hypercholesterolemia, long-standing smoking and left ventricular hypertrophy.

[0025] An increase in arterial distensibility, or a reduction in arterial stiffness, reduces the risk that an individual develops a pathological condition resulting from reduced arterial distensibility.

[0026] In some contexts, an effective amount of relaxin is an amount that is effective in reducing the

45 risk that an individual develops an associated pathological condition or resulting from reduced arterial compliance at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least in 30%, at least 35%, at least 40%, at least 45% or at least 50%, or more, compared to the risk of developing the condition in the absence of relaxin treatment.

[0027] In general, as discussed above, an effective amount of relaxin is one that is effective in increasing arterial compliance. The term "increase" is used interchangeably herein with "stimulate" and "promote." The Examples provide general guidelines for the effective amounts used in rats. Those skilled in the art can easily determine the effective amounts for use in humans, given the guidelines in the Examples. In general, a dose of relaxin is between 0.1 and 500 µg / kg of body weight per day, between 6.0 and 200 µg / kg

55 body weight per day or between 1.0 and 100 µg / kg body weight per day. For administration to a 70 kg person, the dose range would be between 7.0 µg and 3.5 mg a day, between 42.0 µg and 2.1 mg a day or between 84.0 and 700 µg a day. In some contexts, for administration to a human, an effective dose is between 5 µg / kg of body weight per day and 50 µg / kg of body weight per day, or between 10 µg / kg of body weight per day and 25 µg / kg of body weight per day. The amount of relaxin administered will, of course, depend on the size, sex and weight of the subject and the severity of the disease or condition, the form and scheme of administration, the likelihood of recurrence of the disease, and the judgment of the attending physician. In each case the daily dose can be administered for a period of time, instead of a single bolus, depending on the desired effect and the differences in individual circumstances.

[0028] In some contexts, relaxin is administered to the individual at a predetermined rate of maintenance.

65 a serum concentration of relaxin between 0.01 ng / ml and 80 ng / ml, eg between 0.01 ng / ml and 0.05 ng / ml, between 0.05 ng / ml and 0.1 ng / ml, between 0.1 ng / ml and 0.25 ng / ml, between 0.25 ng / ml and 0.5 ng / ml, between 0.5 ng / ml and 1.0 ng / ml, between 1.0

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ng / ml and 5 ng / ml, between 5 ng / ml and 10 ng / ml, between 10 ng / ml and 15 ng / ml, between 15 ng / ml and 20 ng / ml, between 20 ng / ml and 25 ng / ml, between 25 ng / ml and 30 ng / ml, between 30 ng / ml and 35 ng / ml, between 35 ng / ml and 40 ng / ml, between 40 ng / ml and 45 ng / ml, between 45 ng / ml and 50 ng / ml, between 50 ng / ml and 60 ng / ml, between 60 ng / ml and 70 ng / ml or between 70 ng / ml and 80 ng / ml.

5 Determining effectiveness

[0029] Using any known method it can be determined whether a given formulation of relaxin, or if a given dose of relaxin is effective by increasing arterial compliance, reducing arterial stiffness or increasing arterial elasticity. Arterial stiffness can be measured by various methods known to those skilled in the art, including the methods discussed in the Examples.

[0030] A measure of global arterial compliance is the Acarea value, which is calculated from the diastolic decay of the aortic pressure wave [P (t)] using the area method (Liu et al. (1986) Am. J. Physiol. 251: H588-H600), as described in the Example, below. Another measure of global arterial compliance is calculated as the

Pulse rate volume ratio (Chemla et al. (1998) Am. J. Physiol. 274: H500-H505), as described in the Example, below.

[0031] Local arterial compliance can be determined by measuring the elasticity of an arterial wall at a particular point using invasive or non-invasive means. See, e.g., U.S. Patent No. 6,267,728. Regional distensibility, the ducal describes distensibility in an arterial segment, can be calculated from volume and arterial distensibility, and is measured mainly with the use of the pulse wave velocity. See, eg, Ogawa et al., Cardiovascular Diabetology (2003) 2:10; Safar et al., Arch Mal Coer (2002) 95: 1215-18. Other suitable methods of measuring arterial compliance are described in the literature, and any known method can be used. See, eg, Cohn, J.N., "Evaluation of Arterial Compliance", in: Hypertension Primer, Izzo, J.L. and Black, H.R., (eds.), Pub. by

25 Council on High Blood Pressure Research, American Heart Association, pp. 252-253, (1993); Finkelstein, S.M., et al., "First and Third-Order Models for Determining Arterial Compliance", Journal of Hypertension, 10 (Suppl. 6,) S11-S14, (1992); Haidet, G.C., et al., "Effects of Aging on Arterial Compliance in the Beagle", Clinical Research, 40, 266A, (1992); McVeigh, G.E., et al., "Assessment of Arterial Compliance in Hypertension", Current Opinion in Nephrology and Hypertension, 2, 82-86, (1993).

Agonists of the relaxin receptor.

[0032] As used herein, the terms "relaxin preceptor agonist (sic)" and "relaxin" are used interchangeably to refer to biologically active H2 relaxin polypeptides (also referred to herein)

"Pharmaceutically active") from recombinant or native sources (eg, of natural origin); relaxin polypeptide variants, such as amino acid sequence variants; synthetic relaxin polypeptides; and non-peptide relaxin receptor agonists, eg, a relaxin mimetic.

[0033] Biologically active relaxin of natural origin can be derived from human, murine (ie, rat or mouse), swine, or other sources of mammals. The term "relaxin" includes preprorrelaxin H2, prorrelaxin and relaxin; recombinant human relaxin (rhRLX). The amino acid sequences of human relaxin are described in the art. For example, the amino acid sequences of human relaxin are found under the following GenBank Inclusion Nos .: NP_604390 and NP_005050, human H2 prorelaxin. The term "relaxin receptor agonist" includes a human relaxin derived from any of the

45 sequences mentioned above.

[0034] The term "relaxin receptor agonist" also includes a relaxin polypeptide comprising the A and B chains that have N-and / or C- truncation. For example, in H2 relaxin, chain A can vary from A (124) to A (10-24) and chain B from B (-1-33) to B (10-22).

[0035] Also included within the scope of the term "relaxin receptor agonist" are H2 relaxin polypeptides comprising insertions, substitutions or deletions of one or more amino acid residues, glycosylation variants, non-glycosylated relaxin, organic and inorganic salts , covalently modified derivatives of relaxin, preprorrelaxin and prorrelaxin. Also included in the term is a relaxin analog

55 which has an amino acid sequence that differs from a wild sequence (eg, of natural origin), including, but not limited to, relaxin analogues disclosed in the U.S. Patent No. 5,811,395, and U.S. Patent No. 6,200,953. Other suitable relaxins and relaxin formulations can be found in the U.S. Patent No. 5,945,402. A modified relaxin polypeptide is also included to increase the half-life in vivo, eg, PEGylated relaxin (i.e., relaxin conjugated with a poly-ethylene glycol), etc.

[0036] Possible modifications to the amino acid residues of the relaxin polypeptide include acetylation, formylation or similar protection of free amino groups, including amidation of the N-terminal or Cterminal groups, or the formation of esters of hydroxyl or carboxyl groups, eg, modification of tryptophan residue (Trp) in B2 by the addition of a formyl group. The formyl group is a typical example of an easily removable protective group. Other possible modifications include the replacement of one or more of the natural amino acids in the B and / or A chains with a different amino acid (including the D-form of a natural amino acid), including, but not

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limited to, the replacement of the Met fraction in B24 with norleucine (Nle), valine (Val), alanine (Ala), glycine (Gly), serine (Ser) or homoserine (HomoSer). Other possible modifications include the deletion of a natural amino acid from the chain or the addition of one or more extra amino acids to the chain. Additional modifications include amino acid substitutions at the B / C and C / A junctions of prorrelaxin, whose modifications facilitate the cleavage of the

5 prorrelaxin C chain; and the variant relaxin comprising an unnatural C-peptide, e.g., as described in U.S. Patent No. 5,759,807.

[0037] Also included in the term "relaxin receptor agonist" are fusion polypeptides comprising a relaxin polypeptide and a heterologous polypeptide. A heterologous fusion partner polypeptide (eg, a polypeptide that was not relaxin) can be C-terminal or N-terminal to the relaxin portion of the fusion protein. Heterologous polypeptides include immunologically detectable polypeptides (eg, "epitope tags"); polypeptides capable of generating a detectable signal (eg, green fluorescent protein, enzymes such as alkaline phosphatase and others known in the art); therapeutic polypeptides, including, but not limited to, cytokines, chemokines and growth factors.

[0038] Such variations or alterations in the structure of the relaxin molecule resulting in variants are included within the scope of this invention as long as the functional (biological) activity of the relaxin is maintained. In general, any modification of the amino acid sequence or relaxin structure nor increases its immunogenicity in the individual treated with the relaxin variant. Those relaxin variants that have the functional activity described can be easily identified using the methods discussed here.

RELAXIN H2 FORMULATIONS

[0039] The relaxin formulations suitable for use in the methods of the invention are formulations

Pharmaceuticals comprising a therapeutically effective amount of pharmaceutically active relaxin, and a pharmaceutically acceptable excipient. The formulation in some contexts is injectable and in some contexts it is designed for intravenous injection.

[0040] Any known relaxin formulation can be used in the methods of the present invention, as long as the relaxin is pharmaceutically active. "Pharmaceutically active" relaxin is a form of relaxin that results in increased arterial compliance when administered to an individual.

[0041] Relaxin can be administered as a polypeptide, or as a polynucleotide comprising a sequence encoding relaxin. The relaxin suitable for use in the methods of the present invention can be isolated from

From natural sources, it can be synthesized chemically or enzymatically, or produced using standard recombinant techniques known in the art. Examples of methods for making recombinant relaxin are found in several publications, including, eg, Patent Nos. 4,835,251; 5,326,694; 5,320,953; 5,464,756; and 5,759,807.

[0042] The relaxin suitable for use includes, but is not limited to, human relaxin, recombinant human relaxin, relaxin derived from non-human mammals, such as swine relaxin, and any of a variety of variants known in the art. Relaxin, pharmaceutically active variants of relaxin and pharmaceutical formulations comprising relaxin are known in the art. See, e.g., U.S. Patent Nos. 5,451,572; 5,811,395; 5,945,402; 5,166,191; and 5,759,807, related to relaxin formulations, and for teachings related to relaxin production. In the Examples described here, recombinant human relaxin (rhRLX) is identical in the

Amino acid sequence to the natural product of the human H2 gene, consisting of a A chain of 24 amino acids and a B chain of 29 amino acids.

[0043] Relaxin can be administered to an individual in the form of a polynucleotide comprising a nucleotide sequence encoding relaxin. Nucleotide sequences encoding relaxin are known in the art, any of which can be used in the methods described herein. See, eg, Inclusion in Gen Bank No. NM_005059. The relaxin polynucleotides and polypeptides of the present invention can be introduced into a cell with a genetic delivery vehicle. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1: 51-64; Kimura (1994) Human Gene Therapy 5: 845-852; Connelly (1995) Human Gene Therapy 1: 185-193; and Kaplitt (1994) Nature Genetics 6: 148

55 153). The gene therapy vehicles for the administration of constructs that include a coding sequence of a polynucleotide of the invention can be administered locally or systemically. These constructs can use viral or non-viral vector approaches. The expression of such coding sequences can be induced using endogenous or heterologous mammalian promoters. The expression of the coding sequence can be constitutive or regulated.

[0044] The present invention may employ recombinant retroviruses that are constructed to carry or express a selected nucleic acid molecule of interest. Retroviral vectors that can be employed include those described in EP 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/11230; WO 93/10218; Vile and Hart (1993) Cancer Res. 53: 3860-3864; Vile and Hart (1993) Cancer Res.

65 53: 962-967; Ram et al. (1993) Cancer Res. 53: 83-88; Takamiya et al. (1992) J. Neurosci. Res. 33: 493-503; Baba et al. (1993) J. Neurosurg. 79: 729-735; U.S. Patent No. 4,777,127; and EP 345,242.

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[0045] Packaging cell lines suitable for use with the viral vector constructs described above can be easily prepared (see PCT publications WO 95/30763 and WO 92/05266), and used to create producer cell lines (also called lines Vector Cells) for particle production

5 recombinant vectoras. Packaging cell lines are made from human parental cell lines (such as HT1080 cells) or mink, thus allowing the production of recombinant retroviruses that can survive their inactivation in human serum.

[0046] Genetic delivery vehicles can also employ parvoviruses such as adeno-associated virus (AAV) vectors. Representative examples include the AAV vectors shown by Srivastava in WO 93/09239, Samulski et al. (1989) J. Vir. 63: 3822-3828; Mendelson et al. (1988) Virol. 166: 154-165; and Flotte et al. (1993) Proc. Natl Acad. Sci. USA 90: 10613-10617.

[0047] Also of interest are adenoviral vectors, eg, those described by Berkner, Biotechniques (1988)

15 6: 616-627; Rosenfeld et al. (1991) Science 252: 431-434; WO 93/19191; Kolls et al. (1994) Proc. Natl Acad. Sci. USA 91: 215-219; Kass-Eisler et al. (1993) Proc. Natl Acad. Sci. USA 90: 11498-11502; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655.

[0048] Other gene delivery vehicles and methods may be employed, including condensed polycationic DNA bound or not to dead adenovirus alone, for example Curiel (1992) Hum. Gene Ther. 3: 147-154; Ligand bound DNA, for example, see Wu (1989) J. Biol. Chem. 264: 16985-16987; eukaryotic cells of cell delivery vehicles; deposition of photopolymerized hydrogel materials; manual genetic transfer particle gun, as described in the U.S. Patent No. 5,149,655; ionizing radiation as described in the U.S. Patent No. 5,206,152 and in WO 92/11033; neutralization of nucleic charge or fusion with cell membranes.

25 Additional approaches are described in Philip (1994) Mol. Cell Biol. 14: 2411-2418, and in Woffendin (1994) Proc. Natl Acad. Sci. 91: 1581-1585.

[0049] Naked DNA can also be used. Illustrative methods of naked DNA introduction are described in WO 90/11092 and in the U.S. Patent No. 5,580,859. The collection efficiency can be increased using biodegradable latex beads. Latex beads coated with DNA are efficiently transported to the cells after the onset of endocytosis by the beads. The method can be further improved with the treatment of the beads to increase hydrophobia and thus facilitate disruption of the endosome and release of DNA in the cytoplasm. Liposomes that can act as genetically administered vehicles are described in the U.S. Patent No. 5,422,120; PCT Nos. WO 95/13796, WO 94/23697, and WO 91/14445; and EP No. 524 968.

[0050] Non-viral administration suitable for use includes mechanical administration systems such as the approach described in Woffendin et al. (1994) Proc. Natl Acad. Sci. USA 91: 11581-11585. Also, the coding sequence and its expression product can be administered through the deposition of photopolymerized hydrogel materials. Other conventional methods for genetic administration that can be used for coding sequence administration include, for example, the use of the manual genetic transfer particle gun, as described in U.S. Patent No. 5,149,655; the use of ionizing radiation to activate the transferred gene, as described in the U.S. Patent No. 5,206,152 and PCT No. WO 92/11033.

[0051] By using relaxin to increase arterial compliance, any mode can be used

45 pharmaceutically acceptable administration. Relaxin can be administered alone or in combination with other pharmaceutically acceptable excipients, including solid, semi-solid, liquid or aerosol dosage forms, such as, for example, tablets, capsules, powders, liquids, gels, suspensions, suppositories, aerosols, etc. . Relaxin can also be administered in sustained or controlled release dosage forms (eg, using a bioerodible slow release delivery system), including depot injections, osmotic pumps (such as the Alzet implant made by Boost), pills, patches transdermal and transcutaneous (including electrotransport), etc., for prolonged administration at a predetermined rate, preferably in unit dose forms suitable for single administration of precise doses.

[0052] In some contexts, relaxin is administered using an implantable system of administration of

55 medication, eg, a system that is programmable to provide the administration of relaxin. Illustrable programmable implantable systems include implantable infusion pumps. Illustrative implantable infusion pumps, or useful equipment in connection with such pumps, are described, for example, in the U.S. Pat. No. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687; 6,475,180; and 6,512,954. A further illustrative device that can be adapted in the present invention is the Synchromed infusion pump (Medtronic).

[0053] The compositions will typically include a conventional pharmaceutical carrier or excipient and relaxin. In addition, these compositions may include other active agents, carriers, adjuvants, etc. Generally, depending on the intended mode of administration, the pharmaceutically acceptable composition will contain between 65 0.1% and 90%, between 0.5% and 50% or between 1% and 25%, by weight of relaxin, the rest being suitable pharmaceutical carriers, carriers, etc. Actual methods of preparing such dosage forms are known, or clear, for

image8

subject matter experts; for example, see Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1995, or its most recent edition. The human relaxin formulations described in the U.S. Pat. No. 5,451,572 are non-limiting examples of suitable formulations that can be used in the methods of the present invention.

[0054] Parenteral administration is generally characterized by injection, be it subcutaneous, intradermal, intramuscular or intravenous, or subcutaneous. Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Suitable excipients are, for example, water, physiological solution, dextrose, glycerol, ethanol, etc. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, etc., such as, for example, sodium acetate, monolaurate. of sorbitan, triethanolamine oleate, cyclodextrins, etc.

[0055] The percentage of relaxin contained in such parenteral compositions depends greatly on its specific nature, as well as the needs of the subject. However, percentages of active ingredient from 0.01% to 10% in solution are used, and will be higher if the composition is a solid that will be subsequently diluted to the previous percentages. In general, the composition will comprise 0.2-2% of the relaxin in solution.

[0056] Parenteral administration may employ the implantation of a slow-release or sustained-release system, so that a constant dose level is maintained. Several matrices (eg, polymers, hydrophilic gels, etc.) are known in the art to control sustained release, and to progressively decrease the release rate of active agents such as relaxin. See, e.g., U.S. Pat. No. 3,845,770 (describing elementary osmotic pumps); U.S. Pat. Nos. 3,995,651,4,034,756 and 4,111,202 (describing the osmotic pumps in

25 miniature); U.S. Pat. Nos. 4,320,759 and 4,449,983 (describing osmotic multi-chamber systems referred to as osmotic pressure button and pressure-melt pumps); and the U.S. Pat. No. 5,023,088 (describing osmotic pumps with the pattern for sequential dispensing of several dose units).

[0057] Medication release kits suitable for use in the administration of relaxin according to the methods of the invention can be based on any of a variety of modes of operation. For example, the drug release equipment may be based on a diffusive system, a convective system, or an erodible system (eg, an erosion-based system). For example, the medicament release equipment may be an osmotic pump, a steam pressure pump, or an osmotic overflow matrix, e.g., where the medicament is incorporated into a polymer and the polymer provides the release of the formulation of

35 medication concomitant with the degradation of a polymeric material impregnated with the medication (eg, a biodegradable polymeric material impregnated with the medicament). In other contexts, the drug release equipment is based on an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.

[0058] Medication release kits based on a mechanical or electromechanical infusion pump are also suitable for use with the present invention. Examples of such equipment include those described in, for example, the U.S. Pat. No. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, the present treatment methods can be accomplished using any of a variety of non-interchangeable refillable pump systems. Osmotic pumps have been widely described in the literature. See, e.g.,

45 WO 97/27840; and U.S. Pat. No. 5,985,305 and 5,728,396.

[0059] Relaxin can be administered for a period of hours, days, weeks, months or years, depending on several factors, including the degree of arterial stiffness, etc. For example, relaxin is administered for a period of time between about 2 hours to about 8 hours, between about 8 hours to about 12 hours, between about 12 hours to about 24 hours, between about 24 hours to about 36 hours, between about 36 hours to about 72 hours, between about 3 days to a week, between a week to about 2 weeks, between about two weeks to 1 month, between 1 month to about 3 months, between about 3 months to about 6 months, between about 6 months to about 12 months or between 1 year to several years. Administration can be constant, eg, constant infusion for a period of hours, days, weeks, months, years, etc. Alternatively, the administration may be intermittent, e.g., relaxin may be administered once a day for a period of days,

55 once per hour for a period of hours or any other scheme deemed suitable.

[0060] Relaxin formulations can also be administered to the respiratory tract as a nasal or pulmonary inhalation spray or nebulizing solution, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. . In such a case, the particles of the formulation may advantageously have diameters of less than 50 micrometers, preferably less than 10 micrometers.

COMBINATION THERAPIES

[0061] In some contexts, a subject method is modified to include the administration of at least one additional therapeutic agent. Additional therapeutic agents suitable include, but are not limited to, estrogen receptor modulators; antihypertensives such as calcium channel blockers, endothelin receptor antagonists, angiotensin-I converting enzyme (ACE) inhibitors,  adrenergic blockers, vasodilators, diuretics,  adrenergic blockers, renin inhibitors, and angiotensin receptor antagonists ; natriuretic peptides (eg, atrial natriuretic peptide, cerebral natriuretic peptide, and peptide

image9

5 natriuretic type C); agents to block the production of cholesterol (eg, statins) and agents used to treat diabetes.

Estrogen receptor modulators

[0062] Suitable estrogen receptor modulators include any of a variety of estrogenic compounds, as well as Selective Estrogen Receptor Modulators ("SERMs"). SERMs include, but are not limited to, tamoxifen, raloxifene, droloxifene, idoxifene, lasofoxifene, CP-336,156, GW5638, LY353581, TSE-424, LY353381, LY117081, toremifene, fulvestrant, 4- [7-2,2-dimethyl -1-oxopropoxy-4-methyl-2- [4- [2-1-piperidinyl) ethoxy] phenyl] 2H-1-benzopyran-3-yl] -phenyl-2,2-dimethylpropanoate, 4,4'-dihydroxybenzophenone -2,4-dinitrophenyl hydrazone, and SH646.

[0063] Suitable estrogenic compounds include, but are not limited to, mestranol, an estradiol ester, poliestriol phosphate, estrone sulfate, natural estrogens, synthetic estrogens, conjugated estrogens, estradiol, estradiol sulfamates, estradiol valerate, estradiol benzoate, ethinyl estradiol, estrone, estriol, estriol succinate and conjugated estrogens, conjugated equine estrogen, estrone sulfate, 17-estradiol sulfate, sulfate

20 of 17-estradiol, equiline sulfate, 17-dihi-droequiline sulfate, 17-dihydroequiline sulfate, equilenine sulfate, 17-dihydroequilenine sulfate and 17-dihydroequilenine sulfate.

[0064] Also suitable for use are the micronized forms of estrogens, such as micronized estradiol, micronized estradiol sulfamates, micronized estradiol valerate, micronized estradiol benzoate, estrone

Micronized or micronized estrone sulfate or mixtures thereof, notably micronized estradiol, micronized estradiol valerate, micronized estradiol benzoate or micronized estradiol sulfamates.

[0065] Effective doses of estrogens are conventional and well known in the art. Typical approximate doses for oral administration are, eg, ethinyl estradiol (0.001-0.030 mg / day), mestranol 5-25

30 mcg / day), estradiol (including 17-beta estradiol), (0.5-6 mg / day), polystriol phosphate (2-8 mg) and conjugated estrogens (0.3-1.2 mg / day). Doses for other means of administration will be apparent to those skilled in the art. For example, transdermal doses will vary according to the adsorption efficiency of the vehicle used.

[0066] Estrogenic compounds can be administered in any of a variety of conventional ways, including, eg, oral (eg, solutions, suspensions, tablets, dragees, capsules or pills), parenteral (including subcutaneous injection or intravenous, intramuscular or intrasternal injection or infusion techniques), inhalation spray, transdermal, rectal or vaginal administration (eg, by vaginal rings or creams).

40 Antihypertensive Agents

[0067] Suitable ACE inhibitors include, but are not limited to, benazepril (Lotensin®), captopril (Capoten®), enalapril, enalaprilat, fosinopril (Monopril®), lisinopril (Zestril®; Prinivil®), pentopril, quinapril (Accupril®), quinaprilat, Ramipril (Altace®), trandolapril (Mavik®), zofenopril, moexipril (Univasc®), perindopril

45 (Coversyl®; Aceon®), Vasotec®, cilazapril (Inhibace®).

[0068] Suitable diuretics include, but are not limited to, acetazolamide; amiloride; bendroflumethiazide; benzthiazide; bumetanide; chlorothiazide; chlorthalidone; cyclothiazide; ethacrynic acid; furosemide; hydrochlorothiazide; hydroflumethiazide; indacrinone (racemic mixture, or as the (+) or (-) enantiomer alone, or a manipulated ratio,

50 eg, 9: 1 of said enantiomers, respectively); metolazone; methiclotiazide; muzolimine; polythiazide; quinetazone; sodium ethacrylate; sodium nitroprusside; ticrinaphene; triamterene; and trichlormethiazide.

[0069] Suitable β-adrenergic blocking agents include, eg, dibenamine; phentolamine; phenoxybenzamine; prazosin; prazosin / polythiazide (Minizide®); tolazoline; doxazosin (Cardura); terazosin (Hytrin®); tamsulosin

55 (Flomax®); and fuzosima (Uroxatral®).

[0070] -adrenergic blocking agents include, but are not limited to, Betapace (sotalol), Blocadren (timolol), Brevibloc (esmolol), Cartrol (carteolol), Coreg (carvedilol), Corgard (nadolol), Inderal (propranolol ), Inderal-LA (propranolol), Kerlone (betaxolol), Levatol (penbutolol), Lopressor (metoprolol), Normodyne (labetalol), Sectral

60 (acebutolol), Tenormin (atenolol), Toprol-XL (metoprolol), Trandate (labetalol), Visken (pindolol), and Zebeta (bisoprolol).

[0071] Suitable vasodilators include, but are not limited to, diazoxide, hydralazine (Apresoline®), minoxidil, nitroprusside (Nipride®), sodium nitroprusside, diazoxide (Hyperstat IV), verapamil and nifedipine.

[0072] Suitable calcium channel blockers include, but are not limited to, amlodipine (Norvasc®), felodipine (Plendil®), nimodipine, isradipine, nicardipine, nifedipine (Procardia®), bepridil (Vascor®), diltiazem (Cardiazem®), and veramapil (Isoptin®; Calan®).

image10

[0073] Suitable angiotensin II receptor blockers or inhibitors include, but are not limited to, saralasin, losartan (Cozaar), cyclosidomine, eprosartan, furosemide (SIC), irbesartan, and valsartan.

[0074] Suitable renin inhibitors include, eg, pepstatin and renin di-or tripeptide inhibitors; enalkrenine; RO 42-5892 (Hoffman LaRoche), A 65317 (Abbott), CP 80794, ES 1005, ES 8891, SQ 34017; a

10 compound as shown in U.S. Patent No. 6,673,931; and the like

[0075] Suitable endothelin antagonists useful in the present invention include, but are not limited to, atrasentan (ABT-627; Abbott Laboratories), Veletri ™ (tezosentan; Actelion Pharmaceuticals, Ltd.), sitaxsentan (ICOS-Texas Biotechnol- ogy), enrasentan (GlaxoSmithKline), darusentan (LU135252; Myogen) BMS-207940 15 (Bristol-Myers Squibb), BMS-193884 (Bristol-Myers Squibb), BMS-182874 (Bristol-Myers Squibb), J-104132 (Banyu Pharmaceutical), VML 588 / Ro 61-1790 (Vanguard Medica), T-0115 (Tanabe Seiyaku), TAK-044 (Takeda), BQ-788 (Banyu Pharmaceutical), BQ123, YM-598 (Yamanouchi Pharma), PD 145065 (Parke-Davis), A-127722 (Abbott Laboratories), A-192621 (Abbott Labora-tories), A-182086 (Abbott Laboratories), TBC3711 (ICOS-Texas Biotechnology), BSF208075 (Myogen), S-0139 (Shiono -gi), TBC2576 (Texas Biotechnology), TBC3214 (Texas 20 Biotechnology), PD156707 (Parke-Davis), PD180988 (Parke-Davis), ABT-546 (Abbott Laboratories), ABT-627 (Abbott Laboratories), SB247083 (GlaxoSmithKline ), SB 209670 (GlaxoSmithKline); and endothelin receptor antagonists discussed in the art, eg, Davenport and Battistini (2002) Clinical Science 103: 15-35, Wu-Wong et al. (2002) Clinical Science 103: 1075-1115, and Luescher and Barton (2000) Circulation 102: 2434-2440. A suitable endothelin receptor antagonist is TRACLEER ™ (bosentan; produced by Actelion Pharmaceuticals, Ltd.).

TRACLEER ™ is a dual oral active endothelin receptor antagonist, and blocks the binding of endothelin to its endothelin A and B receptors. TRACLEER ™ is usually given at a dose of 62.5 mg bid orally for 4 weeks, followed by a maintenance dose of 125 mg bid orally.

[0076] Other suitable antihypertensives include, eg, aminophylline; crypthenamine acetates and tanatos; deserpidine;

Procaine Mememethoxyphilin; pargiline; clonidine (Catapres); methyldopa (Aldomet); reserpine (Serpasil); Guanetidine (Ismelin); and trimetaphan camsylate.

Statins

[0077] Suitable statins include, without limitation, products such as Crestor, Lipitor, Lescol, Mevacor, Pravochol, Zocor and related compounds such as those discussed, eg, in Rev. Port. Cardio. (2004) 23 (11): 1461-82; Curr Vasc Pharmacol. (2003) 3: 329-33.

Therapeutic agents to treat type 1 or type 2 diabetes

[0078] Other agents suitable for use in a combination therapy with relaxin include therapeutic agents for treating type 1 diabetes, and therapeutic agents for treating type 2 diabetes (eg, agents that increase insulin sensitivity) .

45 Insulin

[0079] Therapeutic agents for treating type 1 diabetes include any form of insulin, as long as insulin is biologically active, that is, insulin is effective in reducing blood levels of insulin in an individual responding to insulin. In some contexts, recombinant human insulin ("regular" insulin) or a recombinant human insulin analog is used. In a particular context, the insulin analog is a monomeric form of insulin, eg, human lispro. In some cases, other forms of insulin are used alone or in combination with recombinant human insulin. Insulin that is suitable for use here includes, but is not limited to, regular insulin (Humulin R, Novlin R, etc.), semi-slow, NPH (isophane insulin suspension; Humulin N, Novolin N, Novolin N PenFill, NPH Ilentil II, NPH-N), slow (exceeding zinc insulin; Humulin L, Ilentin II Lens, 55 Lent N, Novolin N), insulin zinc protamine (PZI), ultra slow (zinc insulin suspension, extended; Humulin U Ultra slow) , insulin glargine (Lantus), insulin aspart (Novolog), acylated insulin, monomeric insulin, superactive insulin, hepatoselective insulin, lispro (Humalog ™), and other insulin analogues or derivatives, and mixtures of any of the above. Commonly used mixtures include mixtures of NPH and regular insulin that contain the following percentages of NPH and regular insulin: 70% / 30%, 50% / 50%, 90% / 10%, 80% / 20%,

60 60% / 40%, and the like. Insulin that is suitable for use here includes, but is not limited to, the forms of insulin shown in the U.S. Patents Nos. 4,992,417; 4,992,418; 5,474,978; 5,514,646; 5,504,188; 5,547,929; 5,650,486; 5,693,609; 5,700,662; 5,747,642; 5,922,675; 5,952,297; and 6,034,054; and published applications of PCT WO 00/121197; WO 90/10645; and WO 90/12814. Insulin analogs include, but are not limited to, superactive insulin analogs, monomeric insulins and hepatospecific insulin analogs.

65

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

[0080] The superactive insulin analogs have a high activity on natural human insulin. Accordingly, such insulin can be administered in substantially smaller amounts while obtaining substantially the same effect with respect to the reduction of serum glucose levels. The superactive insulin analogs include, e.g., human insulin 10-aspartic acid-B; human insulin pentapeptide

(B26-B30) AspB10, Tyr325 --carboxamide; human insulin (B26-B30) lugluB10, TyrB25 --carboxamide; B28-30 insulin destripeptide; an insulin with -aminobutyric acid substituted by A13Leu-A14Tyr; and similar insulin analogs of the human insulin formula des (B26-B30) XB10, TyrB25 --carboxamide, in which X is a substituted residue at position 10 of the B chain. These insulin analogs have potencies between 11 and 20 times that of natural human insulin. All insulin analogs described above involve amino acid substitutions along the A or B chains of natural human insulin, which increases the potency of the compound or changes other properties of the compound. Monomeric insulin includes, but is not limited to lispro.

[0081] Insulin derivatives include, but are not limited to, acylated insulin, glycosylated insulin, and the like. Examples of acylated insulin include those shown in the U.S. Patent No. 5,922,675, e.g., insulin derivatized with a C6-C21 fatty acid (e.g., myristic, pentadecyl, palmitic, heptadecyl or stearic acid) in an amino acid-a-glycine, phenylalanine or lysine

Agents that increase insulin sensitivity

[0082] In some contexts, a treatment regimen for treating an individual with type 2 diabetes comprises administering an additional agent that reduces insulin resistance (eg, increases insulin sensitivity). Suitable agents that treat insulin resistance include, but are not limited to, a biguanide such as metformin (eg, administered in an amount of 500mg or 850mg three times a day), fenformin or a salt thereof; a thiazolidinedionic compound such as troglitazone (see, e.g., US Pat. No. 4,572,912), rosiglitazone (SmithKlineBeecham), pioglitazone (Takeda), Glaxo-Welcome's GL-262570, englitazone (CP-68722, Pfizer) or darglitazone (CP -86325, Pfizer, isaglitazone (MCC-555; Mit-subishi; see, e.g., US Pat. No. 5,594,016), reglitazar (JTT-501), L-895645 (Merck), R-119702 (Sankyo / WL), NN-2344, YM-440 (Yamanouchi), Ragaglitazar (NNC 61-0029

or DRF2725; Novo Nordisk), farglitazar (GI262570), tesaglitazar (AZ 242), KRP-297, and the like; and combinations such as Avandamet ™ (rosiglitazone maleate and metformin hydrochloride).

SUBJECTS FIT FOR TREATMENT

[0083] Individuals who are suitable for treatment with a registered method include any individual who has arterial stiffness (or reduced arterial distensibility) and a disorder associated with or resulting from, reduced arterial distensibility, selected from atherosclerosis , coronary heart disease, diastolic dysfunction, familial hypercholesterolemia, left ventricular hypertrophy, arterial stiffness associated with long-standing smoking, arterial stiffness associated with obesity, arterial stiffness associated with age, systemic lupus erythematosus and hypercholesterolemia.

[0084] Particularly suitable for treatment are individuals whose overall arterial compliance is diminished in relation to a healthy individual in a similar situation. Also particularly suitable for treatment are individuals with a local arterial distensibility measured which is diminished in relation to the expected local arterial distensibility in a healthy individual in a similar situation. Individuals with a measured regional arterial compliance which is diminished in relation to that expected in similarly healthy individuals are also particularly suitable for treatment. In some cases, the global, local or regional arterial compliance of an individual at different points in time can be measured and compared to determine if the arterial compliance in that individual is decreasing and approaching levels that indicate that the intervention is appropriate.

[0085] Individuals who are eligible for treatment with a registered method include individuals who have developed, or are at risk of developing, arterial stiffness associated with age. Such individuals include humans who are over 50 years of age, eg, humans who are between 50 and 60 years of age, between 60 and 65 years of age, between 65 years and 70 years of age, between 70 years and 75 years of age, between 75 years and 80 years of age or older.

[0086] Also suitable for treatment with a registered method are perimenopausal women, menopausal women, postmenopausal women, and women who have stopped their periods for reasons not related to age, eg, as a result of surgery (p .ej., hysterectomy, oophorectomy), and thus have developed, or are at risk of developing, arterial stiffness. Such women can be treated with a combination therapy that includes relaxin and estrogen. Such women can also be treated with a combination therapy that includes relaxin, estrogen and an antihypertensive.

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EXAMPLES

[0087] The following examples are intended to provide those skilled in the art with a sample and complete description of how to make use of the present invention, and are not intended to limit the scope of what the inventors consider as their invention nor are they intended to represent that The experiments below are all the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.) but some experimental errors and deviations must be taken into account. Unless otherwise indicated, the parts are parts by weight, the molecular weight is average molecular weight, the temperature is in degrees Celsius and the pressure is at or near atmospheric. Standard abbreviations may be used, eg, bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotides; i.m., intramuscular; i.p., intraperitoneal;

15 s.c., subcutaneous; and the like

Example 1: Effect of relaxin on systemic arterial resistance and compliance

METHODS

Animals

[0088] Long-Evans female rats 12-14 weeks old were purchased from Harlan Sprague-Dawley (Frederick, Maryland USA). They were provided with the PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds Inc., St.

25 Louis, MO USA) and water ad libitum. The rats were maintained in a 12-hour light / dark cycle. The Institutional Committee for Animal Care and Use of the Magee-Womens Research Institute approved all animal procedures.

Surgical preparation

[0089] In summary, the rats were habituated to Nalgene metabolism cages for one week (VWR Scientific Products), followed by habitation to a 7.5 cm harness / spring assembly for another week while in the metabolism cage (Harvard Apparatus , Holliston, MA USA). The animals were adjusted harness under anesthesia with isoflurane. After this period of habitation, the rats were anesthetized with 60 mg / kg

35 ketamine i.m. and 21 mg / kg pentobarbital i.p., and were placed in prone position on a heating bed. After applying 70% ethanol and betadine to all exposed areas of the skin, ampicillin s.c. (0.2 ml of a solution of 125 mg / ml) and atropine s.c. was also administered. (0.075 ml of a 0.4 mg / ml solution). Then a sterile tygon catheter (18 long, 0.015 in ID, 0.030 OD) connected to a syringe containing Ringer's solution, as well as a sterile thermodilution microwave (22 cm long, F # 1.5; Columbus Instruments, Columbus, OH USA). The tygon catheter was subsequently strung through the hole in the harness and then inserted subcutaneously from the midpoint between both shoulders to the small incision behind the ear using an 18-gauge trocar.

[0090] The thermodilution catheter was also strung through the harness assembly and then inserted via

45 subcutaneously from the midpoint between the scapulae to the skin incision in the left costal margin. The pier was then attached to the harness again. The rat was repositioned on its back. A 1.0 cm skin incision was made in the left inguinal region. The external iliac artery was isolated and prepared for catheterization. The thermocouple was inserted subcutaneously leaving the inguinal incision. The thermocouple was then inserted into the external iliac artery in the rostral direction, so that it easily passed into the internal iliac artery and subsequently into the aorta. At 4.0 cm, the thermocouple was approximately 1.0 cm below the left renal artery. Then a 2.0 cm horizontal incision was made over the trachea, 1.0 cm above the cricoid notch.

[0091] Through this incision, a subcutaneous pocket was dissected in the neck and over the left shoulder. Then the right jugular vein and carotid artery were isolated and prepared for catheterization, the latter facilitated

55 by placing a small roll of gauze under the neck to raise this deep structure. Using the 18-gauge trocar, the tygon catheter was inserted subcutaneously from the small incision behind the right ear outside the neck incision. The tygon catheter was implanted in the right jugular vein at 3.0 cm, thus placing the tip of the catheter at the confluence of the anterior vena cava and the right atrial appendage. The battery / transmitter of a sterile mouse pressure catheter (TA11PA-C20; ca. F # 1.2; Data Sciences International, St. Paul, MN USA) was inserted into the subcutaneous pocket. The mouse pressure catheter was then implanted in the right carotid 2.8 cm, thus placing the tip of the catheter at the confluence of the right carotid and the aortic arch. All wounds were closed with 4-0 silk or autoclips. After instilling 0.05 ml of a solution of heparin in the jugular catheter and connecting it with a straight pin, the rat was placed in the metabolism cage and ampicillin was given in the drinking water for 2 days (100 mg / 50 ml with 2 tablespoons dextrose) The spring and the catheters coming out of the top of the cage are

65 assured.

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[0092] Terbutrol was administered s.c. for post-operative analgesia as soon as the rats recovered sufficiently from anesthesia. For the administration of low doses of recombinant human relaxin (4.0 µg / h rhRLX) for 10 days, two Alzet model 2002 osmotic mini-pumps (Durect Corporation, Curpertino, CA USA) were inserted subcutaneously into the back of the animal under anesthesia with isoflurane . For dose administration

5 discharges for 10 days (25 µg / h), an Alzet model 2ML2 osmotic pump was implanted. After completing the measurement for the last time point, the rat was anesthetized with 60 mg / kg pentobarbital i.v. Blood was obtained from the abdominal aorta for rhRLX, osmolality and hematocrit levels. The position of the jugular catheter in relation to the right atrium, the placement of the pressure catheter in relation to the aortic arch and the position of the thermocouple in relation to the left renal artery were recorded.

In vivo studies: Hemodynamics and systemic arterial mechanical properties

[0093] First time control studies were conducted in 5 rats, to document the stability of systemic hemodynamics in a period of 17 days after surgery. The measurements were recorded on days 4-5,

15 7-8, 9-10,13-14, and 16-17 after surgery. The protocols with low and high doses of rhRLX included 6 and 7 rats, respectively. In addition, the vehicle for rhRLX (20mM sodium acetate, pH 5.0) was administered to another 6 rats. After 2 baseline measurements of systemic hemodynamics on days 5 and 7 after surgery, the low or high dose of rhRLX was administered per osmotic pump. Systemic hemodynamics was evaluated again on days 3, 6, 8 and 10 days after starting infusion of rhRLX or vehicle.

[0094] Each measurement consisted of 4 to 6 records of cardiac output and blood pressure waves that were obtained when the rat was sleeping or resting. At least 10 minutes were allowed between records. These measurements were obtained between 9am and 3pm.

25 [0095] Cardiac output. To measure cardiac output, we use the thermodilution technique. Osborn et al. (1986) Am. J. Physiol. 251: H1365-H1372. Ringer's solution of known volume and temperature was injected into the anterior vena cava using the Micro Injector 400 (Columbus Instruments). Cardiac output was calculated from the change in blood temperature (Cardiotherm 400R, Columbus Instruments). The cardiac output determined by the Cardiotherm 400R was calculated as:

[0096] CO = [(BT-IT) * VI] / ƒBT (t) where BT is the blood temperature (recorded by the thermocouple implanted in the abdominal aorta), IT is the injected temperature (room temperature), VI is the volume of the injected (150 µL), and Bt (t) is the blood temperature as a function of time.

35 [0097] Blood pressure. Instant aortic pressure was recorded using a blood pressure telemetry system (Data Sciences International, St. Paul, MN USA). Mills et al. (2000) J. Appl. Physiol 88: 1537-1544. Aortic pressure was recorded with a pressure catheter implanted in the aortic arch through the right carotid and transmitted to an external receptor. The steady state aortic pressure was digitized online using a PC-based data acquisition system with 16-bit resolution and 2000 Hz sample rate and stored as text files for offline analysis. Each measurement consisted of a sample duration of 30 seconds.

[0098] Aortic pressure analysis. The analysis of the acquired data and calculations of glonal CA was performed with a computerized program adapted and developed using MATLAB software (MathWorks Inc., Natick, MA USA). In summary, individual beats (3 -15 cycles) of the 10 seconds of the aortic pressure record were selected,

45 immediately preceding the measurement of cardiac output. The assembly was averaged as described by Burattini et al. ((1985) Comput. Biomed. Res. 18: 303-312) to yield a single representative beat for each beat. The mean arterial pressure (MAP), peak systolic pressure (Ps) and end-of-diastole pressure (Pd) were calculated from this average beat. Pulse pressure (PP) was calculated as Ps-Pd. Systemic vascular resistance (SVR) was calculated by dividing the MAP by CO.

[0099] Global arterial distensibility. Two measures of global arterial compliance were calculated. The first (Acarea) was calculated from the diastolic decay of the aortic pressure wave [P (t)] using the area (2) method: ACarea = Ad / [SVR (P1-P2)] where P1 and P2 are pressures at the beginning and end of the decay curve

diastolic, respectively, and Ad is the area under the P (t) wave in this region. The second measure of compliance

The overall arterial blood pressure was calculated as the pulse volume beat-pressure pressure (Chemla et al. (1998) Am. J. Physiol. 274: H500-H505). The beat volume was defined as CO / HR.

In vitro studies: Passive arterial mechanics

[0100] Non-pregnant female rats were administered rhhRLX (4 µg / h) or vehicle per osmotic minobombra for 5 days. A kidney was removed and placed in cold HEPES buffered physiological solution (PSS, a modified Kreb buffer). The physiological solution HEPES was composed of (in mmol / L): sodium chloride 142, potassium chloride 4.7, magnesium sulfate 1.17, calcium chloride 2.5, potassium phosphate 1.18, HEPES 10, glucose 5.5 and was at pH 7.4 a 37 ° C A dissection stereomicroscope, small forceps and iridectomy scissors were used

65 to isolate the interlobar arteries as described by Gandley et al.

image13

[0101] ((2001) Am. J. Physiol. 280: R1-R7) (internal diameter not pressurized, 100-200 µm). An arterial segment was then transferred to an isobaric arteriograph (Living Systems Instrumentation, Burlington, VT USA and mounted in 2 glass microcannulas suspended in the chamber. After the residual blood was discharged from the lumen of the artery,

5 The distal cannula was occluded to prevent flow. The proximal cannula was attached to a pressure transducer, a pressure servo controller and a peristaltic pump. The servo controller maintained a selected intraluminal pressure that was changed stepwise. An electronic system of analysis of dimensions obtained the measurements of arterial diameter.

[0102] The vessels were incubated in the bath with 10-4 M papaverine and 10-2 M EGTA in calcium-free PSS HEPES. After an equilibrium period of 30 min, the transmural pressure increased in 14 steps starting at 0 mmHg to 150mmHg. The internal and external diameters as well as the wall thickness were measured after each pressure step when the vessel had reached a steady state. The mean radius of the wall (Rm) and the circumferential stress of the wall () were calculated from these data as described above (Cholley et al. (2001) J.

15 Appl. Physiol 90: 2427-2438). The elastic properties of the vascular wall were quantified in terms of the incremental elastic modulus (Einc), which was calculated from the -Rm ratio (Pagani et al. (1979) Circ. Res. 44: 420-429).

Serum measurements

[0103] Serum osmolality was measured using a freezing point depression instrumentation osmometer (Model 3 MO; Advanced Instruments, Needham Heights, MA USA). Serum rhRLX levels were measured with a quantitative sandwich immunoassay as previously described (Jeyabalan et al. (2003) Circ Res. 93 (12): 1249-57).

25 Preparation of rhRLX

[0104] Model 2002 osmotic mini pumps (Durect Corporation, Cupertino, CA USA) were used to administer rhRLX for 10 days at a dose of 4 µg / h which produced circulating relaxin concentrations similar to those measured from the beginning to the middle of pregnancy in rats. that is, 10-20 ng / ml when pregnancy-induced renal vasodilation is maximal in this species. An osmotic model 2ML2 mini-pump was used to administer rhRLX at a dose of 25 µg / h for 10 days, which we expected to produce circulating hormone concentrations comparable to those recorded from mid to late gestation when increases in CO are observed. and decreases in the SVR in this species. The rhRLX (Connetics, Palo Alto, California USA) supplied as a

5.0 mg / ml solution in 20 mM sodium acetate pH 5.0 was diluted in the same buffer.

Statistic analysis

[0105] Data are presented as means + SEM. ANOVA of repeated measures of one or two factors (Zar (1984) Biostatistical Analysis, Englewood Cliffs, NJ: Prentice Hall) was used to compare the values of the means between several groups. If significant principal effect or interactions were observed, comparisons were made between the groups using Fisher's LSD or Dunnett's test. Student's paired ‘t ’test was used to compare compound means during rhRLX infusion (i.e., averaged values at all time points during rhRLX infusion) with baseline. A regression analysis of minimum squares on the relationships was performed

45 of -Rm and Einc-Rm. The analysis of excess variance (or extra sum of squares) was used to compare these relationships between the groups treated with vehicle and with relaxin. A p <0.05 was taken as significant.

RESULTS

In vivo studies

[0106] Time control. The stability of systemic arterial hemodynamics and load over a period of 17 days after surgery in control rats (Table 1). Heart rate declined significantly due to a training effect as previously reported (Conrad and Russ (1992) Am. J. Physiol. 31: R472-477). He

55 beat volume increased reciprocally, so that the CO did not change. The other variables did not change significantly in the period of 17 days after surgery, so this conscious rat model can be used to obtain significant data under the experimental conditions described below (Table 1).

Table 1. Time Control Rats

image14

Time ∆T (° C) CO HR * SV * (mL) ACarea SVR MAP after (bpm) (µl / mmHg) surgery (mL / min) (mmHg.s / mL) (mmHg)

(days)

4-5 0.37 ± 0.01 119 ± 3 428 ± 7 0.28 ± 0.01 6.8 ± 0.3 57 ± 2 107.6 ± 0.8

5 7-8 0.37 ± 0.01 121 ± 3 378 ± 8 0.32 ± 0.01 7.2 ± 0.3 55 ± 2 107.3 ± 1.5 9-10 0.38 ± 0.01 120 ± 4 382 ± 8 0.31 ± 0.01 6.9 ± 0.3 55 ± 2 108.1 ± 1.5 13 -14 0.35 ± 0.01 115 ± 4 354 ± 5 0.32 ± 0.01 7.3 ± 0.3 58 ± 2 107.0 ± 1.5 16-17 0.32 ± 0.02 122 ± 3 349 ± 7 0.36 ± 0.01 8.0 ± 0.4 52 ± 2 103.7 ± 2.2

15 [0107] Mean ± SEM. N = 5 rats. T, change in blood temperature after injection of Ringer's solution into the right heart chambers; CO, cardiac output; HR, heart rate; SV, beat volume; Acarea, global arterial distensibility calculated using the area method; SVR, systemic vascular resistance; MAP, mean blood pressure. * P <0.05 per ANOVA of repeated single-factor measurements.

[0108] Rats given the vehicle (for rhRLX). These results were derived from 3 rats that received the vehicle for rhRLX at the infusion rate of 1 µl / h, and from other 3 rats that received the vehicle for rhRLX at the infusion rate of 5 µl / h. (These correspond to the rates for the administration of low and high doses of rhRLX, respectively.) The results were comparable, and therefore, were combined. Figs. 1 and 2 show the percentage change from baseline for systemic hemodynamics and other variables. Similar to control studies

25 of time, there was a significant decrease in heart rate, which was compensated for an insignificant increase in beating volume, so that the CO remained unchanged. The other variables remained relatively constant. Combining all time points during vehicle administration produced global changes in the CO, global AC and SVR of -1.4 + 1.3, 2.2 + 4.6, and 0.4 + 3.4% (SIC) of the basals, respectively (all p NS vs basals). As expected, there was no measurable rhRLX in the serum, and the osmolality was 309 + (SIC) 6 mOsm / kg water.

[0109] Rats that received low doses of rhRLX (4 mglh). Absolute values for systemic hemodynamics and other parameters are shown in Table 2, while Figs. 1 and 2 show the temporal pattern of percentage change from baseline.

35 Table 2. Rats that received low doses of rhRLX (4mg / h)

Days ∆T (° C) CO * HR SV * (mL) ACrearea * SVR * MAP

after (mL / min) (bpm) (µl / mmHg) (mmHg · s / mL) (mmHg)

of the

minibomb

Baseline 0.34 ± 0.02 128 ± 2 417 ± 6 0.31 ± 0.01 6.2 ± 0.3 51 ± 2 111.6 ± 3.7 2-3 0.31 ± 0.01 151 ± 6 436 ± 9 0.35 ± 0.01 7.2 ± 0.3 43 ± 3 110.2 ± 4.9 45 6 0.31 ± 0.01 149 ± 7 419 ± 60.36 ± 0.02 7.1 ± 0.5 46 ± 3 116.9 ± 3.8

8 0.31 ± 0.01 159 ± 7 427 ± 11 0.37 ± 0.02 7.4 ± 0.5 44 ± 2 112.1 ± 2.8

10 0.31 ± 0.01 153 ± 5 418 ± 10 0.37 ± 0.01 7.7 ± 0.5 44 ± 2 115.2 ± 5.8

[0110] Mean ± SEM. N = 6 rats. Two baseline measurements were made on days 5 and 7 after surgery. These results were averaged for each rat. For abbreviations, see Table 1.

[0111] Low doses of rhRLX significantly increased CO in relation to baseline and infusion of

55 vehicle (Fig. 1A). The infusion of rhRLX prevented the decrease normally observed in the HR (c.f. vehicle, Fig. 1B), and the hormone significantly increased the SV (Fig. 1C). Thus, increases in SV and HR were combined to increase CO in relation to rats that were infused with a vehicle. Systemic vascular hemodynamics fell significantly in relation to baseline and vehicle infusion (Fig. 2A), while MAP remained unchanged (Fig. 2B).

[0112] The global CA increased significantly in relation to baseline and vehicle infusion (Fig. 2C). There was no significant change in pulse pressure; however, the pulse rate beat volume ratio, another index of arterial distensibility, increased significantly during infusion of low doses of rhRLX relative to baseline and vehicle infusion (Fig. 2D). Examine the course over time of changes in variables 65 that showed a significant change with the administration of low doses of rhRLX (i.e., significant value of F

image15

for relaxin and / or interaction). By paired comparisons post hoc data at different time points (Fisher's LSD). The CO and the SV were significantly higher than baseline on day 3. While the SV continued to increase until day 8 (p <0.05, day 8 vs day 3) (Fig. 1C), there were no significant changes over time in the CO beyond day 3 (Fig. 1A). This was the result of a small but insignificant drop in HR from day 3 to

5 day 8 (Fig. 1B).

[0113] The SVR and both measures of global CA were significantly altered on day 3; after that there were no significant subsequent changes (Fig. 2). In general, the maximum changes in arterial hemodynamics and mechanical properties after the administration of low doses of rhRLX were observed at the earliest time point (day 3), without subsequent temporal alterations. Combining all the time points during the administration of low doses of rhRLX produced a global increase in CO and in the global CA of 19.2 + 4.8 and

21.4 + 3.6% above baseline, respectively, and a global decrease in SVR of 15.5 +2.4% below baseline (all p <0.01 vs. baseline). The serum rhRLX and osmolality were 14 + 2 ng / ml and 284 + 2 mOsm / kg water, respectively. The latter decreased significantly compared to vehicle infusion.

15 [0114] Rats that received high doses of rhRLX (25 mg / h). Absolute values for systemic hemodynamics and other variables are presented in Table 3, and Figs. 1 and 2 show the percentage change from baseline. The results for the infusion of high doses were comparable to the administration of low doses in the direction, but somewhat smaller in magnitude.

Table 3. Rats that received high doses of rhRLX (25 µg / h)

Days ∆T (° C) CO * HR SV (mL) ACarea * SVR * MAP after (mL / min) (µl / mmHg) on (bpm) (mmHg.s / mL) (mmHg) minipump

0.37 ± 0.02 129 ± 6 438 ± 10 0.30 ± 0.02 7.7 ± 0.7 53 ± 3 111.6 ± 3.7 base line

2-3 0.33 ± 0.02 141 ± 7 454 ± 13 0.31 ± 0.02 8.5 ± 0.8 49 ± 4 110.2 ± 4.9

6 0.35 ± 0.02 147 ± 4 432 ± 10 0.34 ± 0.01 8.1 ± 0.4 48 ± 2 116.9 ± 3.8

8 0.35 ± 0.03 150 ± 5 451 ± 9 0.33 ± 0.01 9.4 ± 0.7 45 ± 2 112.1 ± 2.8

10 0.34 ± 0.02 146 ± 9 442 ± 6 0.33 ± 0.02 9.2 ± 1.0 48 ± 3 115.2 ± 5.8

35 [0115] Mean6SEM. N = 7 rats. Two baseline measurements were made on days 5 and 7 after surgery. These results were averaged for each rat. For abbreviations, see Table 1.

[0116] Temporal analysis of changes in individual variables with high doses of rhRLX was performed similarly to low doses of rhRLX. Once again, CO (Fig. 1A), SV (Fig. 1C), SVR (Fig. 2A) and global AC (SV / PP method) (Fig. 2D) were maximally altered at the earliest time point examined (day 3), without other significant changes later. The temporal response of the global CA calculated by the area method (Fig. 2C) deviated slightly from this general pattern - the ACarea on day 6 was no different from the baseline. This is probably an aberrant measurement because the second measure of global CA at all time points (Fig. 2D) and ACarea on days 45, 3, 8, and 10 (Fig. 2C) were significantly greater than baseline. Combining all the time points during the administration of high doses of rhRLX produced a global increase in CO and the global CA of 14.1 + 3.2 and

15.6 + 4.7% above baseline, respectively, and a global decrease in SVR of 9.7 + 2.4% below baseline (all p <0.02). The serum relaxin and osmolality were 36 + 3 ng / ml and 287 + 1 mOsm / kg of water, respectively. The latter decreased significantly compared to vehicle infusion.

[0117] Waves of blood pressure. Representative arterial waves of a single basal rat and after administration of rhRLX are shown in Fig. 3A. They illustrate that the mouse pressure catheter (TA11PA-C20) provides high fidelity records necessary to determine the global CA. The assembly of the average blood pressure waves, derived using the methodology proposed by Burattini et al. (supra) are shown in

Fig. 3B for the 3 groups of rats on day 10 of infusion. As discussed above, the SV increased significantly and the SVR decreased significantly after administration of rhRLX (Tables 2 and 3). If these were the only alterations, we would expect to see a clear change in the pressure wave morphology: increased pulse pressure and a hurried diastolic decay of blood pressure. However, as illustrated in Fig. 3B, the administration of rhRLX did not significantly affect the morphology of the blood pressure wave, as indicated by the unchanged pulse pressure and diastolic decay. This invariable pressure wave morphology, in the presence of an increased SV and a decreased SVR, is consistent with a simultaneous increase in global CA.

In vitro studies

65 [0118] Passive arterial mechanics. These in vitro experiments were performed to examine the effects of rhRLX administration on the passive mechanical properties (i.e., in the absence of an active smooth muscle tone) of the vascular wall. As mentioned before (Methods section), the primary measurements consisted of the inner and outer diameters of the cases at various levels of intraluminal pressure. The circumferential stress of the wall (σ) and mean radius of the wall (Rm) were calculated from these primary measurements and the σ-Rm ratio was used to quantify the elastic behavior of the vessel wall (eg, incremental elastic modulus , Einc). The

image16

5 ratios of σ-Rm (Fig. 4A) and Einc-Rm (Fig. 4B) for the minor renal arteries were significantly different between the two groups (p <0.001 by analysis of excess variance) so that σ and Einc were minors for a given Rm in the group treated with relaxin. In contrast, the stress-free Rm, Rmo (i.e., Rm at o = 0), was not different between the two groups (treated with relaxin: 10565 mm; treated with vehicle: 9866 mm). Thus, the axis Rm can be considered as the circumferential tension of the wall. These data indicate that treatment with relaxin significantly reduced the stiffness of the vascular wall (Einc) to payable values of MRI (tension). This reduced passive stiffness of the wall contributes to the high overall CA seen in conscious animals treated with relaxin (vide supra).

Example 2: Effects of relaxin on systemic arterial hemodynamics and mechanical properties in 15 conscious rats: sex dependence and dose response

METHODS

Animals

[0119] Male and female Long-Evans rats 12-14 weeks old were purchased from Harlan Sprague-Dawley (Frederick, Maryland USA). They were provided with the PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds Inc., St. Louis, MO USA) and water ad libitum. The rats were kept in a light-dark cycle of 12:12 h. This research is in accordance with the Guide for the care and use of laboratory animals published by the

25 US National Institute of Health (NIH Publication No. 85-23, revised in 1996).

Administration of recombinant human relaxin (rhRLX)

[0120] RhRLX (BAS, Palo Alto, California USA) was provided as a 5.0 mg / ml solution in a buffer (20 mM sodium acetate, pH 5.0). It was diluted when necessary in the same buffer. For the low dose infusion protocol, two model 2002 osmotic mini pumps (Durect Corporation, Cupertino, CA USA) were used to administer rhRLX for 10 days at the dose of 4 mg / h. This dose was designed to produce circulating relaxin concentrations similar to those measured during the onset to the middle phase of pregnancy in rats, that is, 10-20 ng / ml (Sherwood OD, Endocrinol Rev 25 (2): 205- 234, 2004). For the high dose infusion protocol 35, an osmotic model 2ML2 mini pump was used to administer rhRLX at 50 µg / h for 6 days, which is expected to produce serum concentrations comparable to those recorded at the end of pregnancy (Sherwood OD, Endocrinol Rev 25 (2): 205-234, 2004) when increases in CO and decreases in SVR are observed in this species (Gilson et al., Am J Physiol 32: H1911-H1918, 1992; Slangen et al., Am J Physiol 270: H1779-1784, 1996). Finally, in a third protocol, rhRLX was administered by bolus i.v. for 3 min (13.4µg / ml) followed by an infusion

i.v. Continue for 4 hours.

Surgical preparation

[0121] As in Example 1, the rats were anesthetized for 60 mg / kg ketamine i.m. and 21 mg / kg pentobarbital i.p.

45 They were then instrumented, using a sterile technique, as follows: (i) a tygon catheter implanted in the right jugular vein with the tip at the junction of the anterior vena cava and right atrium, (ii) a thermodilution microsonde (36 cm long, F # 1.5; Columbus Instruments, Columbus, OH USA) implanted in the abdominal aorta through the left femoral artery with the tip 1.0 cm below the left renal artery, and (iii) a pressure catheter from mouse (TA11PA-C20, F # 1.2; Data Science Inter-national, St. Paul, MN USA) implanted in the right carotid with the tip at the junction of the right carotid and the aortic arch. For acute administration of rhRLX, another tygon catheter was implanted in the inferior vena cava through the left femoral vein so that the tip was 1.0 cm below the right renal artery.

[0122] After instilling 0.05 ml of a solution of heparin in the jugular catheter and connecting it to a straight pin, the

55 rats received ampicillin in the drinking water for 2 days (100 mg / 50 m1 (SIC) with 2 tablespoons of dextrose). Terbutrol s.c. was administered. for postoperative analgesia.

[0123] For chronic administration of low doses of recombinant human relaxin (4.0 µg / h rhRLX) in male rats for 30 days, two 2002 model Alzet osmotic mini-pumps (Durect Corporation, Curpertino, CA USA) were inserted subcutaneously into the animal's back under anesthesia with isoflurane. For chronic administration of high doses in female rats for 6 days (80 µg / h), an Alzet model 2ML2 osmotic mini pump was implanted. High doses of rhRLX were also administered to another group of female rats acutely by intravenous bolus for 3 min (13.4 µg / ml) followed by a continuous infusion for 4h.

65 [0124] After completing the measurement for the last time point, the rats were anesthetized with 60 mg / kg pentobarbital i.v. Blood was obtained from the abdominal aorta for measurements of plasma rhRLX levels.

5

fifteen

25

35

Four. Five

55

65

The position of the jugular catheter in relation to the right atrium, the placement of the pressure catheter in relation to the aortic arch and the position of the thermocouple in relation to the left renal artery were recorded.

Hemodynamics and systemic arterial mechanical properties

[0125] The low and high dose rhRLX protocols included 7 male and 9 female rats, respectively. After two baseline measurements of systemic hemodynamics on days 5 and 7 after surgery, low or high doses of rhRLX were administered by the osmotic pump. Systemic hemodynamics was reassessed on days 3, 6, 8 and 10 after starting the relaxin infusion for male rats at low doses and on days 3 and 6 for female rats at high doses. Each measurement consisted of 4 to 8 records of cardiac output and blood pressure waves obtained when the rat was sleeping or resting. Seven to 10 minutes were allowed between the records. These measurements were obtained between 9 AM and 3 PM. [0126] For acute administration of high doses of rhRLX, 5 female rats were used. Baseline measurements of systemic hemodynamics were obtained followed by intravenous infusion of high doses of rhRLX for 4 hours. Systemic hemodynamics was continuously evaluated during the 4 hours of infusion.

[0127] We use the thermodilution technique (Osborn et al., Am JPhysiol 251: H1365-H1372, 1986) to measure cardiac output. Instantaneous aortic pressure waves were recorded using a blood pressure telemetry system (Data Sciences International, St. Paul, MN USA) (Mills et al., JAppl Physiol 88: 1537-1544, 2000). The aortic pressure recorded by the pressure catheter implanted in the aortic arch was transmitted to an external receptor. The steady state aortic pressure was digitized online using a PC-based data acquisition system with 16 bits of resolution and a sample rate of 2000 Hz and stored as text files for offline analysis. Each measurement consisted of a sample duration of 30 seconds.

[0128] The analysis of the acquired data and the calculation of the global CA was performed using an adjustable computer program developed using Matlab software (MathWorks Inc., Natick, MA USA). In summary, individual beats (3 -15 cycles) of the 10 seconds of the aortic pressure record were selected, immediately preceding the measurement of cardiac output. The assembly was averaged as described by Burattini et al. (2) to produce a single representative beat for each beat. The mean arterial pressure (MAP), peak systolic pressure (Ps) and end-of-diastole pressure (Pd) were calculated from this averaged beat. Pulse pressure (PP) was calculated as Ps-Pd. Systemic vascular resistance (SVR) was calculated by dividing the MAP by CO.

[0129] Two measures of global arterial compliance were calculated. The first (ACarea) was calculated from the diastolic decay of the aortic pressure wave [P (t)] using the area method (18):

image17

where P1 and P2 are the pressures at the beginning and at the end of the diastolic decay curve, respectively, and Ad is the area under the P (t) wave curve in this region. The second measure of global arterial distensibility was calculated as the pulse-beat volume ratio, SV / PP (Chemla et al., Am J Physiol 274: H500-H505, 1998). The beat volume was defined as CO / HR.

Serum measurements

[0130] Serum osmolality was measured using a freezing point depression instrumentation osmometer (Model 3 MO; Advanced Instruments, Needham Heights, MA USA). Serum rhRLX levels were measured with a quantitative sandwich immunoassay as previously described (Jeyabalan et al., Circ Res 93: 1249-1257,2003).

Statistic analysis

[0131] Data are presented as means ± SEM. For comparison, data from a previous study are included (Conrad et al., Endocrinology 145 (7): 3289-3296,2004; Example 1) where low and medium doses of rhRLX were administered to female rats. ANOVA of repeated measures of two factors (Zar JH, Biostatistical Analysis. Englewood Cliffs: Prentice Hall, 1984) was used to compare the mean values between male and female rats with low doses at various time points. The same analysis was performed to compare mean values between low, medium and high doses of rhROX in female rats at various time points. ANOVA of repeated measures of one factor (Conrad et al., Endocrinology 145 (7): 3289-3296,2004) was used to compare the mean values at several time points after starting the acute infusion of high doses of rhRLX with the baseline values. If significant main effects or interactions were observed, paired comparisons were made between the groups using Fisher's LSD test. Student's paired ‘t ’test was used to compare the mean compound values (defined below) during chronic rhRLX infusion with baseline. A p <0.05 was taken as significant. Finally, linear regression was used to analyze the relationships between the magnitudes of the change in each arterial property of the individual rats in response to the infusion of relaxin and the baseline values of that property. Group differences in linear regression parameters were examined using ANCOVA, implemented as multiple linear regression with dummy variables (Gujarati D, Am Statistician 24: 18-22, 1970).

image18

RESULTS

5 [0132] Male rats that received low doses of rhRLX (4 µg / h). The temporal patterns of several systemic hemodynamic variables, expressed as a percentage of baseline values, are illustrated in Fig. 5 and the absolute values of these variables are presented in Table 4. For comparison purposes, the data from our previous study. (Conrad et al., Endocrinology 145 (7): 3289-3296,2004) examining the effects of rhRLX infusion at 4 µg / h in female rats are also presented in Fig. 5. The low dose of rhRLX increased significantly CO in relation to baseline in male rats. There was a slight increase (~ 6%), but statistically significant, in HR in male rats treated with relaxin (Fig. 5A). However, there was a greater increase in the SV (Fig. 5B) indicating that the elevation in the CO resulted primarily from an increase in the SV, and to a lesser extent by an increase in the HR. The mean blood pressure did not change significantly during

15 infusion of rhRLX (Fig. 5D). At the final time point (i.e., day 10 after the start of rhRLX infusion), there was no statistically significant difference between the effects of rhRLX administration on systemic hemodynamics in male and female rats.

[0133] The temporal effects of rhRLX infusion on systemic arterial properties in male rats, expressed as a percentage of baseline values, are shown in Fig. 6. Again, the absolute values for these variables are presented in Table 4 and the data of female rats are also shown in Fig. 6 for comparison. Systemic vascular resistance fell significantly in relation to baseline (Fig. 6A), while both measures of arterial compliance (ACarea and SV / PP) increased significantly (Figs. 2B and 2C). At the end time point (i.e., day 10 after the start of rhRLX infusion), changes in the

25 arterial properties were not statistically different between male and female rats.

Table 4. Male rats that received low doses of rhRLX (4mg / h)

Days later ∆T (° C) CO * HR SV * (mL) * SVR * MAP

ACarea

minipump (mL / min) (bpm) (µl / mmHg) (mmHg.s / mL) (mmHg)

Line of 0.32 ± 0.03 148 ± 9 416 ± 12 0.36 ± 0.03 7.5 ± 0.6 49 ± 3 115.8 ± 2.4 base

3 0.31 ± 0.01 160 ± 6 440 ± 8 0.36 ± 0.01 7.6 ± 0.5 46 ± 1 121.3 ± 3.6

6 0.29 ± 0.02 169 ± 4 441 ± 6 0.38 ± 0.01 7.9 ± 0.3 43 ± 1 119.9 ± 1.7

8 0.27 ± 0.02 178 ± 6 445 ± 4 0.40 ± 0.01 8.7 ± 0.4 41 ± 1 118.8 ± 2.0

10 0.28 ± 0.02 183 ± 11 442 ± 5 0.41 ± 0.02 8.8 ± 0.5 41 ± 2 121.6 ± 2.6

35 [0134] Mean ± SEM. N = 7 rats. ∆T, change in body temperature after injection of Ringer's solution into the right heart cavities; CO, cardiac output; HR, heart rate; SV, heartbeat volume, ACarea, global arterial compliance calculated using the area method; SVR, systemic vascular resistance; MAP, mean blood pressure. * P <0.05 per ANOVA of repeated single-factor measurements.

[0135] We calculated a compound mean change from baseline for each variable by averaging the values at all successive time points during the infusion of rhRLX that were characterized by a significant change from baseline and were not significantly different from each other ( that is, the set phase). This produced global increases in CO and global CA of 20.5 6 4.2% and 19.4 6 6.9% from baseline, respectively, and a global decrease in SVR of 12.7 6 3.9% from baseline (all p < 0.05 vs. baseline). There was no statistical difference between these results in male rats and those reported for female rats (Example 1). The serum rhRLX was 17.7 6 1.1 ng / ml, a value similar to that previously observed in female rats that received the same rhRLX regimen, 14.0 6 2.0 ng / ml.

55 [0136] Female rats that received high doses of rhRLX (50 µg / h). The absolute values of systemic hemodynamics and arterial properties are listed in Table 5 and their temporal patterns after starting rhRLX infusion are shown in Figs. 3 and 4. For comparison purposes, the data in Example 1 examining the effects of low dose (serum concentration = 14 6 2 ng / ml) and medium (serum concentration = 36 6 3 ng / ml) of rhRLX infusion in female rats they are also shown in Figs. 3 and 4. Infusion of low and medium doses of rhRLX significantly increased CO, mainly increasing SV. Both doses also significantly reduced SVR and increased CA (Conrad et al., Endocrinology 145 (7): 3289-3296,2004). These alterations were observed at the earliest time point studied -3 days after the start of rhRLX administration. The serum concentration of rhRLX for high dose infusion in the present study was

71.5 6 1.6 ng / ml. However, there was no change from baseline in any of the systemic hemodynamics or

65 arterial properties (Figs. 3 and 4). Thus, the effects of rhRLX on systemic hemodynamics and arterial properties are apparently biphasic.

image19

Table 5. Female rats that received high doses of rhRLX (50mg / h)

Days ∆T (° C) CO HR SV (mL) ACarea SVR MAP *

after (mL / min) (bpm) µ / mmHg) (mmHg.s / mL) (mmHg) the minipump

Baseline 0.34 ± 0.02 134 ± 5 425 ± 14 0.32 ± 0.02 7.0 ± 0.4 53+ 2 115.9 ± 2.7

3 0.32 ± 0.02 141 ± 6 438 ± 6 0.32 ± 0.01 7.2 ± 0.3 52 ± 2 120.2 ± 3.3

6 0.31 ± 0.01 146 ± 6 437 ± 8 0.33 ± 0.02 7.4 ± 0.4 51 ± 3 121.1 ± 2.8

[0137] Media6SEM. N = 8 rats. ∆T, change in blood temperature after injection of Ringer's solution into the right heart chambers; CO, cardiac output; HR, heart rate; SV, beat volume; ACarea, global arterial compliance calculated using the area method; SVR, systemic vascular resistance; MAP, mean blood pressure. * P <0.05 per ANOVA of repeated single-factor measurements.

[0138] To determine if there would be significant alterations in systemic hemodynamics and arterial properties in response to high doses of rhRLX treatment at a time point before 3 days after

At the start of administration of rhRLX, 5 additional conscious female rats were treated with i.v. infusion. Acute rhRLX for 4 hours. The serum concentration of rhRLX was 64.1 ± 1.0 ng / ml. The temporal effects of the short-term high dose of rhRLX infusion on systemic hemodynamics and arterial properties in female rats, expressed as a percentage of baseline values, are shown in Figs. 5 and 6. The cardiac frequency increased significantly (~ 13%) at the 2 and 4 hour time points (Fig. 9A). This increase in HR was offset by a decrease (although statistically insignificant) in the SV (Fig. 9B), resulting in no significant change in CO (Fig. 9C). There was a small increase (~ 8%), but statistically significant, in the MAP (Fig. 9D). There were no statistically significant changes from baseline in any of the systemic arterial properties (Fig. 9).

[0139] The data described above suggest that the magnitude of the change in arterial properties of individual rats (male or female) in response to infusion of low doses of rhRLX depended on the baseline value of that particular property. To validate this trend, the relationship between baseline SVR, ACarea and SV / PP and their respective compound percentage changes from baseline during rhRLX infusion was analyzed. Linear regression analysis revealed that the effect of rhRLX infusion (i.e., percentage change from baseline) on SVR (Fig. 9A) and CA, measured by ACarea (Fig. 9B) and SV / PP (Fig. 9C), depended highly on their baseline values. Specifically, rats with low baseline CA were characterized by a greater increase in CA in response to relaxin treatment. Similarly, rats that had baseline SVR responded to relaxin with a greater decrease in SVR. Subsequent analysis (ANCOVA) indicated that these linear relationships were not different between male and female rats.

[0140] The previous results show that relaxin induces similar effects on systemic hemodynamics and arterial properties in male and female rats, although relaxin is traditionally considered to be a female hormone and is not believed to circulate in male rats (Sherwood OD , Endocrinol Rev 25 (2): 205-234, 2004).

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Claims (11)

image 1  Claims 5 1. Relaxin H2 for use in a method to increase arterial compliance in a human being, where the subject has a decrease in global arterial compliance in relation to global arterial compliance in a healthy subject, where the subject has one or more conditions selected from the group consisting of: atherosclerosis, coronary heart disease, diastolic dysfunction, hypercholesterolemia, left ventricular hypertrophy, arterial stiffness associated with long-standing smoking, arterial stiffness associated with obesity, associated arterial stiffness 10 with age and systemic lupus erythematosus. 2. Relaxin H2 for use in a method to increase arterial compliance in a human being, where the subject has a decrease in global arterial compliance in relation to the overall arterial compliance in a healthy subject, where the subject has one or more conditions selected from the group consisting of: atherosclerosis, 15 coronary disease, diastolic dysfunction, hypercholesterolemia, left ventricular hypertrophy, arterial stiffness associated with long-standing smoking, arterial stiffness associated with obesity, arterial stiffness associated with age, and systemic lupus erythematosus. 3. Relaxin H2 for use in a method to increase arterial compliance in a human being, where the 20 subject has a decrease in global arterial distensibility in relation to global arterial distensibility in a healthy subject, where the subject has one or more conditions selected from the group consisting of: atherosclerosis, coronary heart disease diastolic dysfunction, hypercholesterolemia, left ventricular hypertrophy, arterial stiffness associated with long-standing smoking, arterial stiffness associated with obesity, arterial stiffness associated with age, and systemic lupus erythematosus. 25

Four.

The H2 relaxin for use according to statement 1, where the overall arterial compliance is measured from the diastolic decay of the aortic pressure wave using the area method.

5.

Relaxin H2 for use according to statement 1, where the overall arterial compliance is calculated

30 as the heartbeat volume-pulse pressure ratio, and where said heartbeat volume is defined as the heart rate ratio at heart rate. 6. Relaxin H2 for use according to statement 3, where regional arterial compliance is measured using the pulse wave velocity. 35 7. The H2 relaxin for use according to any preceding statement, where the H2 relaxin is administered to the subject at a predetermined rate to maintain a serum H2 relaxin concentration between 0.5 and 80 ng / ml. 8. The H2 relaxin for use according to any preceding statement, where the H2 relaxin is recombinant 40 H2 relaxin. 9. The H2 relaxin for use according to any preceding statement, where the H2 relaxin is administered daily. 45 10. The relaxin H2 for use according to any preceding statement, where the relaxin H2 is administered in an injectable formulation, a sustained release formulation or a formulation administrable by continuous infusion. 11. The H2 relaxin for use according to any preceding statement, where the subject is a premenopausal woman, a menopausal woman or a postmenopausal woman. 12. Relaxin H2 for use according to any preceding statement, where the postmenopausal woman has stopped her period as a result of hysterectomy or oophorectomy. 55 22